JPH0767651A - Type ii interleukin-1 acceptor - Google Patents

Abstract

Type II IL-1 receptor (type II IL-1R) proteins, DNAs and expression vectors encoding type II IL-1R, and processes for producing type II IL-1R as products of recombinant cell culture, are disclosed. <IMAGE>

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates generally to cytokine receptors, and more particularly to interleukin-1 receptors.

[0002]

2. Description of the Related Art Interleukin-1α (IL-1α)
And interleukin-1β (IL-1β) are distantly related polypeptide hormones that play a central role in immune regulation and the inflammatory response. These two proteins act on various cells and have multiple biological activities. The difference in biological activity attributable to IL-1α and IL-1β is due to specific plasma membrane receptors that bind to both IL-1α and IL-1β. IL-1α and I
Due to the widespread biological activity by L-1β, I
It was originally believed that the L-1 receptor must be highly conserved among various species and expressed in a variety of cells.

As a result of structural evaluation by the ligand affinity cross-linking technique, IL-1α and IL-1β are the same cell surface receptor molecules of T cells and fibroblasts, despite remarkable differences in sequence. (Dower et al., Nature (Lond
on) 324: 266, 1986; Bird eta.
l. , Nature (London) 324: 263,
1986; Doweret al. , Proc. Nat
l. Acad. Sci. USA 83: 1060, 19
86). From cDNA expression cloning and N-terminal side sequence analysis as an integral membrane glycoprotein having a molecular weight of 80,000 kDa, which binds to IL-1α and IL-1β,
IL-1 receptors on murine and human T cells have been identified (Sims et al. Science).
241: 581, 1988; Sims et al., Proc. Na
tl. Acad. Sci. USA 86; 8946, 19
89).

However, at present, this 80 kDa
It has been shown that the IL-1 receptor protein does not convey all of the various biological potencies of IL-1. Further affinity cross-linking analysis was performed using the Epstein Barr virus (Epstein B
arr virus (EBV) -transformed human B cell lines VDS-O and 3B6, EBV-positive bar kit (B).
urkitt's) lymphoma cell line Raji,
And suggests that the IL-1 receptor in the pre-murine B cell line 70Z / 3 has a molecular weight of 60,000 to 68,000 kDa (Matsushima et al.
ima et al. J. Immunol. 136:
4996, 1986; Bensimon et al.
et al. , J. Immunol. 142: 22
90, 1989; Bensimon et al. Immunol.
143; 1168, 1989; Holuk et al.
et al. J. Biol. Chem. 262: 1
6275, 1987; Chizonite
e) et al. , Proc. Natl. Acad. Sci. U
SA86: 8029, 1989; Bomszik et al. (B
omsztyk et al. , Proc. Natl.
Acad. Sci. USA 86: 8034, 198.
9). Furthermore, when comparing the biological characteristics and kinetic analysis of the IL-1 receptor of the Raji B cell line and the EL-4 murine T lymphoma cell line, Raji cells showed that It has been found to have low binding affinity but to have a higher density of receptors per cell (Horuk et al., J. Biol. Chem. 26.
2: 16275, 1987). Raji cells also. IL-
1 could not be absorbed and showed different receptor binding affinities for IL-1 analogues (Holk et al., J. B.
iol. Chem. 262: 16275, 1987).
These data indicate that the IL-1 receptor expressed on B cells (referred to herein as the type IIIL-1 receptor) is the IL-1 receptor found on T cells and other cell types (here type (Referred to as IIL-1 receptor).

To analyze the structural and biological properties of type II IL-1R and the role of type II IL-1R in the response of various cell populations to IL-1 stimulation, or in treatment, diagnosis. In order to effectively use type II IL-1R in analyses, a homogeneous composition is required. Such compositions can theoretically be obtained by purification of the receptor expressed in cultured cells, or by cloning and expression of the gene encoding the receptor. However, before the present invention, there were some obstacles and the object was not achieved.

First, a cell line that constitutively and continuously expresses type II IL-1R at a high level has not been previously known, and a cell line that is known to express type II IL-1R. Is a small number (500 to 2,000
Expression of only receptors / cells hampered efforts to obtain amino acid sequence information and to purify the receptor in sufficient quantities to generate monoclonal antibodies. Due to the small number of receptors, practical cloning analysis-based cloning methods have also been impossible.

Next, type I IL-R and type II I
Cross-hybridization with the murine type IL-1R cDNA was also not possible due to the significant differences in the DNA sequence of L-1R (Bomstic (Bo
msztyk) et al., Proc. Natl. Acad, S
ci. USA 86: 8034, 1989, and Chizzonite et al. , Proc. Nat
l. Acad. Sci. USA 86: 8029, 198.
9).

Third, even if a protein composition that was sufficiently purified to allow N-terminal protein sequencing was obtained, the degeneracy of the genetic code obtained from it was further Without a great deal of experimentation, a suitable probe could not be determined. Many iterative trials will be required to determine the probe with the specificity necessary to determine the hybridizing sequence in the cDNA library. Direct expression cloning techniques avoid the need for repeated screens with different probes of unknown specificity and allow other receptors (eg type IIL-
1R), but a cDNA library obtained from cells expressing a small number of type II IL-1Rs,
Not sensitive enough to be used to immobilize the -1R clone.

Therefore, efforts to purify type II IL-1R and to clone and express the gene encoding type II IL-1R lacked the purified receptor, an appropriate source of receptor mRNA. And lack of a cloning technique that is sufficiently sensitive.

[0010]

The present invention provides a purified, homogenous type II IL-1R protein and a type I IL-1R protein.
Isolated DNA encoding the IL-1R protein
The sequences are provided, particularly human type II IL-1R or analogs thereof. Such a DNA sequence is preferably (a) a cDNA clone having a nucleotide sequence derived from the coding portion of the native type II IL-1R gene, such as clone 75; (b) under mildly stringent conditions. IL- which has a biological activity and can hybridize with the cDNA clone of (a).
A DNA sequence encoding the 1R molecule; and (c) (a)
And selected from the group consisting of DNA sequences that code for a IL-1R molecule that is degenerate and biologically active as a result of the genetic code of the DNA sequence defined in (b). The present invention also provides a recombinant expression vector comprising a DNA sequence as defined above, a recombinant type II IL-1R molecule produced using the recombinant expression vector, and a recombinant type II IL utilizing the expression vector. A method of producing a -1R molecule is provided.

The present invention also provides a protein composition comprising a substantially purified, homogeneous protein and type II IL-1R.

The present invention also relates to therapy, diagnosis, type II.
Provided is a composition comprising an effective amount of soluble native or recombinant receptor protein prepared according to the methods described above for use in the analysis of IL-1R or the production of antibodies against type IIIL-1R.

These and other aspects of the invention will be apparent upon reference to the following detailed description.

The definition "IL-1" refers to IL-1α and IL-1β together.

"Type II interleukin-1 receptor" and "type II IL-1R" are capable of binding interleukin-1 (IL-1) molecules and are naturally associated with proteins such as mammalian plasma membrane proteins. When taking the structure of
It refers to a molecule that plays a role in transmitting a signal from L-1 to a cell. Mature full-length human type II IL-1R is a glycoprotein with an apparent molecular weight of approximately 60-68 kDa. Specific examples of type II IL-1R proteins include S
It is shown in EQ ID NO: 1 and SEQ ID NO: 12. As used herein, the above terms include analogs or subunits of the native type II IL-1R protein that have IL-1 binding or signaling activity. In particular, a truncated form of the type II IL-1R protein and a soluble form are also included, as defined below. In the absence of species designation, type II IL-1R refers generically to mammalian type II IL-1R, including but not limited to human, murine, bovine type II IL-1R. Similarly, if there is no special designation for the deletion mutation, type I
The term I IL-1R refers to type II IL-1R
All forms of type II IL-1R, including biologically active variants or analogs thereof. "Interleukin-1 receptor" or "IL-1R" means type
IIL-1 receptor and type II IL-1 receptor are collectively referred to.

"Soluble type II IL-1R" as used in the context of the present invention is a protein, or a substantially equivalent analogue thereof, which contains all or part of the extracellular portion of native type II IL-1R. Are substantially similar and secreted from cells, but have the ability to bind IL-1 or inhibit the signaling of IL-1 through the cell surface bound IL-1R protein. Refers to something. Soluble type II IL-1R protein may comprise part of the transmembrane region if it can be secreted from the cell. Soluble type II
Specific examples of IL-1R proteins include SEQ ID N
A protein having an amino acid sequence of 1-330 or 1-333 of O: 1, and 1- of SEQ ID NO: 12
It contains proteins with 342 and 1-345 amino acid sequences. Inhibition of IL-1 signaling activity can be expressed in primary cells or by expressing endogenous IL-1R and reacting biologically with IL-1, or by introducing recombinant IL-1R DNA.
It can be determined using a cell line that is biologically responsive to L-1. The cells are contacted with IL-1 and the metabolic effects caused thereby are analyzed. If affected by the action of the ligand, the recombinant receptor has signaling activity. A typical method for determining whether a polypeptide has signaling activity is the Izelda (Idze
rda) et al. J. Exp. Med. 171: 861
(1990); Curtis et al. , Pro
c. Natl. Acad. Sci. USA 86: 304
5 (1989); Pryues et al. , E
MBO J. 5: 2179 (1986) and Ko (Ch
ou) et al. J. Biol. Chem. 262: 184
2 (1987).

The term "isolated" or "purified" when used in the context of this specification to define the purity of a type II IL-1R protein or protein composition Alternatively, it means that the protein composition is substantially free of contamination with other, naturally occurring or endogenously derived proteins, and the contaminating protein residue in the production process is 1% or less. Such compositions, however, may include other proteins as stabilizers, carrier vehicles or cotherapeutic agents. Type IIIL-1
R is "isolated" if it is detectable as a single band when silver is stained on a polyacrylamide gel.

When the term "substantially similar" is used in defining an amino acid or nucleic acid sequence, a unique dependent sequence, eg a variant sequence, is derived from the original sequence.
It has been altered by one or more substitutions, deletions, additions, and its total effect is, for example, type II IL-
In the 1R binding assay, it is meant to retain the determined biological activity of the type II IL-1R protein, as described below in Example 5. Alternatively, the nucleic acid subunit or analog is (a) D
NA sequence is SEQ ID NO: 1 or SEQ ID
If it is obtained from the code area of NO: 12, (b)
If the DNA sequence is under mild stringent conditions (2
5% formamide, 42 ° C., 2 × SSC), or alternatively, under more stringent conditions (50
% Formamide, 50 ° C., 2 × SSC, or 50% formamide 42 ° C., 2 × SSC), which is capable of hybridizing to the DNA sequence of (a) and has biological activity.
If it encodes an L-1R molecule, or
A DNA sequence resulting from the genetic code for the DNA sequence defined in (a) or (b), which encodes a biologically active IL-1R molecule, is defined by the specific DNA sequence disclosed herein. “Substantially similar”.

As used herein, "recombinant" means that the protein is obtained in a recombinant (eg microbial or mammalian) expression system. “Microbial” refers to recombinant proteins made in bacterial or fungal (eg, yeast) expression systems. As a product, "recombinant microbial" defines a protein that is essentially free of natural endogenous substances and is free of natural glycosylation. Most bacterial cultures such as E. The protein expressed in E. coli does not contain glycans. Proteins expressed in yeast may undergo a different pattern of glycosylation than that expressed in mammalian cells.

"Biological activity" as used throughout this specification as a characteristic of type II IL-1R is a measurable amount of IL-1, preferably at least type II.
0.01 nmole of I per 1 nmole of IL-1R
Sufficient amino acid sequence similarity to bind L-1 is SEQ ID NO: 2 or SEQ ID NO: 13.
To a cell or to a molecule that shares sufficient amino acid sequence similarity to allow IL-1 stimulation to be transduced into cells, eg, as part of a hybrid receptor structure. Means a molecule. Further, preferably within the scope of the present invention, a biologically active type II IL-
The 1R is capable of binding 0.1 nmoles or more of IL-1 per nmole of receptor, and most preferably 0.5 nmoles of IL-1 per nmole of receptor.

"DNA sequence" refers to a polymer of DNA, which is a discrete fragment or part of a larger DNA structure, at least once in substantially pure form, ie, endogenous. Nucleotide sequence derived from DNA isolated by a standard biochemical technique using a cloning vector, for example, derived from DNA isolated in a form free from contamination of substances and having a quantity capable of identifying, manipulating and recovering the sequence Part of. Such sequences are desirably provided in a form with an open reading frame uninterrupted by an endogenous untranslated region or intron characteristically present in eukaryotic genes. However, it will be apparent that genomic DNA containing the appropriate sequence may also be used.
The sequence of untranslated DNA is located 5'or 3'to the open reading frame, where it does not interfere with manipulation or expression of the coding region.

"Nucleotide sequence" refers to a hetylopolymer of deoxyribonucleotides. The DNA sequence encoding the protein provided by the present invention is cDNA
Assembled from fragments and short oligonucleotide linkers or a series of oligonucleotides to provide synthetic genes that can be expressed in recombinant transcription units.

A "recombinant expression vector" is (1) a single or a plurality of genetic factors that control gene expression, such as a promoter or enhancer, and (2) transcribed into mRNA and translated into protein. , Structural or
Refers to a plasmid containing a transcription unit containing a collection of coding sequences, (3) appropriate transcription and translation initiation and termination sequences. Structural factors for use in yeast expression systems desirably include a leader sequence that enables the protein translated by the host cell to be secreted extracellularly. Instead,
Recombinant proteins expressed without a leader or transport sequence may contain an N-terminal methionine residue. This residue may optionally be further cleaved from the expressed recombinant protein to provide the final product.

"Recombinant microbial expression system" means, for example, E.
bacteria such as E. coli and S. By a substantially homogeneous monoculture of host microorganisms, such as a yeast such as S. cerevisiae, which has stably integrated a recombinant transcription unit into the chromosomal DNA, or
It has a recombinant transcription unit as one of the constituents of the resident plasmid. In general, the cells that make up the system are the progeny of a single progenitor recombinant. The recombinant expression system as defined herein expresses a heterologous protein by the induction of a regulatory factor linked to the DNA sequence or the synthetic gene to be expressed.

Similar to type II IL-1R protein
The present invention relates to isolated recombinant mammalian type II IL-1.
An R polypeptide is provided. Type II in the present invention
IL-1R proteins are, for example, primates, humans,
Includes murine, canine, feline, bovine, ovine, equine, caprine and porcine type IIIL-1R. Type II I
L-1R is a human or mouse type II IL-1RD
By interspecific hybridization using a single-stranded cDNA obtained from the NA sequence, for example, mammalian cDNA
From the NA library, type II IL-1RcDN
To isolate A, human clone 75 can be used as a hybridization probe and obtained. Like most mammalian genes, mammalian type II ILs
The -1R is probably encoded by multiple exons. A broad range of all-alternative m that is identical or similar to the claimed cDNA, which may result from different post-transcriptional mRNA splicing.
RNA constructs are considered to be within the scope of this invention. The DNA sequence encoding IL-IR-II, presumably in the form of another splicing arrangement, can be isolated from the following cells and tissues: B lymphoblastoid system (CB23, CB33, Raji, RPMI1788, A
RH77), quiescent and especially activated peripheral blood T cells, monocytes, monocyte cell line THP1, neutrophils, bone marrow, placenta, endothelial cells, keratinocytes (especially activated) , And HepG2 cells.

Type II IL-1R within the scope of the present invention
Derivatives of S. cerevisiae also contain various structural forms of the biologically active native protein. Type II IL due to, for example, ionizable amino and carboxyl groups
The -1R protein may be in the form of an acid or basic salt or in a neutral form. Individual amino acid residues may also be modified by oxidation or reduction.

The primary structure of an amino acid forms covalent or pseudo-bonds with other chemical moieties such as sugar chains, lipids, phosphates, acetyl groups, and the like, or creates variant amino acid sequences. It may be modified by Shared inducers represent a particularly functional group of type II ILs.
It is prepared by binding to the amino acid side chain of -1R or the N-terminus or C-terminus. Type II IL within the scope of the present invention
Other derivatives of -1R are type II IL, such as by synthesis in recombinant culture, such as N-terminal or C-terminal fusion.
-1R or a fragment thereof and covalent or pseudo-linkage of other protein or polypeptide. For example, the bound peptide may be a signal (or leader) at the N-terminal portion of the protein that transports the protein, either simultaneously or post-translationally, from its synthetic site to its functional site, inside or outside the cell membrane or wall. ) Polypeptide sequences (eg yeast α-factor leader). Type II IL-1R protein fusions are type II IL
Peptides (eg, polyHis) to facilitate purification and identification of -1R can also be included. Type II IL-
The amino acid sequence of 1R is also the peptide Asp-Tyr-
Lys-Asp-Asp-Asp-Asp-Lys (D
YKDDDDK) (Hopp et al., Bio /
Technology 6: 1204, 1988). The latter sequence is highly antigenic and provides an antibody recognition site for reversible binding of specific monoclonal antibodies, allowing rapid analysis and easy purification of the expressed recombinant protein. This sequence is also
sal enterokinase) by Asp
-It is specifically cleaved immediately after the Lys pair. The fusion protein capped with this sequence is E. It may become resistant to intracellular digestion in E. coli.

The Type II IL-1R derivative is also
Immunogen, receptor-based immunoassay reagents, or
It can also be used as a binding reagent for affinity purification procedures for IL-1 or other binding ligands. Type II
IL-1R derivatives may also be obtained using cross-linking reagents that act on cysteine and lysine residues such as M-maleimidobenzoylsuccinide ester and N-hydroxysuccinide. Type II I
The L-1R protein also includes cyanogen bromide,
By adsorbing various insoluble substrates, such as bisoxylene, carbonyldiimidazole, or tosyl-activated agarose structures, through reactive side groups or on the polyolefin surface (with or without glutaraldehyde crosslinking), May be covalently bound. Type II once bound to substrate
IL-1R is anti-type I (for analytical or purification purposes)
It may be used to selectively bind to the IIL-1R antibody or IL-1.

The present invention also relates to natural glycosylation,
Also included is the type II IL-1R.
Is type II IL-1R expressed in yeast or a mammalian expression system such as COS-7 cells similar in molecular weight and glycosylation type to a natural molecule depending on the expression system? , Or may be slightly different. E. Type II IL-1 in bacteria such as E. coli
Expression of RDNA produces molecules that are not glycosylated. Functional mutant analogs of mammalian type II IL-1R in which the N-glycosylation site is inactivated can be made by oligonucleotide synthesis and ligation or site-directed mutagenesis techniques. These similar proteins can be made in good yields in homogeneous and carbohydrate reduced form using yeast expression systems. The N-glycosylation site of eukaryotic proteins has been characterized by three amino acids, Asn-A ₁ -Z, where A
₁ is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine supplies side chain amino acids for the covalent addition of carbohydrates. An example of an N-glycosylation site of human type II IL-1R is amino acids 66-68,7 of SEQ ID NO: 1.
2-74, 112-114, 219-221 and 277.
-279. Such a site may replace the Asn or Z residue with another amino acid, delete Asn or Z, insert a non-Z amino acid between A ₁ and Z,
It can be reduced by inserting something other than Asn between Asn and A ₁ .

Type II IL-1R derivatives are type I
It can be obtained by mutating I IL-1R or its subunit. Type II, as shown here
The IL-1R variant is similar to the type II IL-1R in polypeptide but is a native type II IL-1.
It has a different amino acid sequence from R due to deletion, insertion and substitution.

A bioequivalent analogue of a type II IL-1R protein may include, for example, various substitutions of residues or sequences, or deletions of residues or sequences not necessary for terminal or internal biological activity. Lost, and may be created by. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges during regeneration. Other means of mutagenesis also include modification of adjacent divalent basic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. In general, the substitution should be conservative. That is, the most preferred amino acid substitution is one that replaces a residue having similar physicochemical properties. Similarly, when attempting a deletion or insertion, the possible effect of the deletion or insertion on the biological activity must be considered. Substantially similar polypeptide sequences generally include a similar number of amino acid sequences, as defined above, but are soluble type II ILs.
The C-terminal truncation to create -1R will contain fewer amino acid residues. Type II IL
The deletions and substitutions are made with similar or conserved substitution sequences to preserve the biological activity of the -1R.
That is, it is desirable that the residue in question be replaced with a biologically similar residue. Examples of saved substitutions are Ile,
Substitution of hydrophobic residues such as compatibility with Val, Leu, or Ala, Lys and Arg, Glu and As
It includes substitutions between polar residues such as n, Gln and Asn. Other conserved substitutions are also well known, for example substitutions of entire regions of similar hydrophobicity.
In addition, human, murine, and other mammal type I
Certain amino acids that differ between the IIL-1Rs suggest additional conservative substitutions that do not alter the essential biological properties of type II IL-1R.

The type II IL-1R subunit can be prepared by deleting the terminal or internal residues or sequences. The present invention relates to, for example, soluble type II corresponding to all or part of the extracellular region of type II IL-1R.
A C-terminal deletion resulting in IL-1R is intended. Desirably the resulting protein is capable of binding IL-1.
A particularly desirable sequence is a sequence in which the transmembrane region and intracellular region of type II IL-1R are deleted or substituted with a hydrophilic residue, which promotes secretion of the receptor into cell culture medium. . Soluble type II IL-1R protein is
If the soluble type II IL-1R protein can be secreted from the cells, it may partially contain the transmembrane region. For example, soluble human type II IL-1R
Contains amino acid sequences 1-333 or 1-330 of SEQ ID NO: 1 and may contain amino acid sequences 1-345 and 1-342 of SEQ ID NO: 12. Alternatively, the soluble type II IL-1R protein may have resulted from the deletion of the C-terminal region of type II IL-1R in the extracellular region that is not required for IL-1 binding. For example, SEQ ID NO: 1 and SEQ
An array of ID NO: 12, 313 and 32, respectively
C-terminal deletions may have been made to make proteins with 5 or more amino acids. These amino acids are cysteines, believed to be necessary for maintaining the tertiary structure of the type II IL-1R molecule, and type II
Allows the IL-1R molecule to bind IL-1. Soluble type II IL-1R structure is type II
The 3'region of the DNA encoding IL-1R was deleted, and the DNA was inserted into an appropriate expression vector for expression. Typical methods for the construction of such soluble proteins are described in Examples 2 and 4. Type II I generated
The L-1R protein is expressed in IL as described in Example 5.
The ability to bind -1 is measured. Both such DNA sequences encoding soluble type II IL-1R and biologically active soluble type II IL-1R proteins resulting from such structures are intended to be included within the scope of the present invention. ing.

Mutations in the nucleotide sequences created for the expression of type II IL-1R analogues naturally preserve the reading frame phase of the coding sequence and appear to negatively affect the translation of the receptor mRNA. M, such as loops and hairpins
It is desirable not to have a complementary region that is hybridizable so as to give rise to a secondary structure of RNA. The site of introduction of the mutation is predetermined, but the nature of the mutation itself need not be predetermined. For example, in order to select the optimum property of mutation at a certain site, random mutagenesis to the target codon was performed, and the expressed mutant type II
IL-1R is screened for those with the desired activity.

Not all mutations in the nucleotide sequence encoding type II IL-1R are expressed as end products. For example, a nucleotide substitution is
It occurs to enhance expression and basically avoid secondary structures such as loops in transcribed mRNA (EPA75,44
4A, included herein by reference) or, for example, the famous E. E. coli for expression of E. coli. It occurs to supply codons that are translated more quickly by the host of choice, such as those preferred by E. coli.

Mutations can be introduced at intended positions by synthetic oligonucleotides containing a mutant sequence flanked by restriction enzyme sites that allow ligation with fragments of the original sequence. After ligation, the resulting reconstructed sequence encodes an analog with the insertion, substitution, or deletion of the amino acid of interest.

Alternatively, a modified gene having a special codon changed to have the necessary substitution, deletion or insertion can be prepared by using a site-directed mutagenesis method using an oligonucleotide. . An exemplary method of making the modifications described above is described by Walder et al., (Gen).
e42: 133, (1986); Boyer (Baue).
r) et al. (Gene 37:73, 1985); Craik (Biotechniques, Jan.
uary1985, 12-19); Smith (Smit
h) et al. (Genetic Engineering: p
rinciples and Methods, Plen
um Press, 1981); and US Pat. Nos. 4,518,584 and 4,73.
No. 7,462 discloses suitable techniques and is incorporated in the reference.

Both monovalent and polyvalent forms of Type II IL-1R are useful in terms of the compositions and methods of this invention. The multivalent form has multiple type II IL-1R binding sites for IL-1 ligand. Eg 2
Valuable soluble type II IL-1R has two extracellular regions of type II IL-1R separated by a linker region.
It has two vertical repeating structures. Other polyvalent forms are also available, for example, a type II IL-1R chemically synthesized using conventional coupling techniques on polymers selected from the group including carriers generally available for clinical use, Ficoll, polyethylene glycol, or dextran. There is also a possibility that a chemically bound substance is produced. Type II instead
When IL-1R is biotin chemically bound, biotin-type II IL-1R then binds avidin,
A tetramer of avidin / biotin / type IIIL-1R molecule results. Type II IL-1R also includes dinitrophenol (DNP) or trinitrophenol (T
NP) and the resulting complex is anti-DNP or anti-T.
Precipitated with NP-IgM to form a decavalent complex with decavalent type II IL-1R binding sites.

Recombinant chimeric antibodies can be prepared by substituting the type II IL-1R sequence with either or both of the heavy chain and the light chain of the immune blobulin molecule and having an unchanged constant region. For example, type II IL-
The 1R / IgG ₁ chimera consists of two chimeric genes-type II IL-1R / human _kappa light chain chimera (type IIIL).
-1R / C _κ ) and type II IL-1R / human _γ 1
It results from a heavy chain chimera (type II IL-1R / C _γ-1 )-. After transcription and translation of the two chimeric genes, the gene product assembles into one chimeric antibody molecule with type II IL-1R and exhibits bivalence. Type II like this
The multivalent form of IL-1R will have an enhanced binding affinity for IL-1 ligand. For further details on the construction of such chimeric antibody molecules, see WO 89/096.
22 and EP 315062.

Expression of Recombinant Type II IL-1R The present invention provides a recombinant expression vector for amplifying or expressing DNA encoding type II IL-1R. Recombinant expression vector is mammalian type II IL
A suitable transcriptional or translational regulatory factor from mammals, microorganisms, viruses or insects, having a DNA fragment of synthetic or cDNA origin, which encodes -1R or a biological analogue thereof.
A operably linked, replicable DNA construct. Transcriptional units are generally (1) single or multiple genetic factors that control gene expression, such as a transcription promoter or enhancer, (2) transcribed into mRNA and translated into protein, structural or , A coding sequence, and (3) suitable transcription and translation initiation and termination sequences, which are described in detail below. Such regulators may include operator sequences that control transcription, ie, sequences that encode suitable mRNA ribosome binding sites. A replication gene in a host, which is usually obtained by an origin of replication, and a selection gene for promoting recognition of transformants are additionally included. DNA regions are operably linked when they are functionally related to each other. For example, a signal peptide (secretory leader) DNA is operably linked to the DNA of a polypeptide if it is expressed as a precursor responsible for secreting the polypeptide. A promoter is a coding sequence if it controls the transcription of the sequence.
Be operably coupled. Alternatively, the ribosome binding site, if added to allow translation,
It is operably linked to a code array. Generally, operably linked means connected in series, and, in the case of secretory leaders, in continuous and open reading frame. Structural elements intended for use in yeast expression systems desirably include a leader sequence that enables the protein translated by the host cell to be secreted extracellularly. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it will contain an N-terminal methionine site. This residue is optionally subsequently cleaved from the expressed recombinant protein to provide the final product.

The DNA sequence coding for mammalian type II IL-1R to be expressed in microorganisms is:
Transcription of DNA into mRNA can be stopped prematurely. However, premature termination of transcription is desirable. For example,
In order to obtain a soluble receptor that does not bind to the cell membrane, for example, when a mutant with an advantageous truncated C-terminal lacking the transmembrane portion is obtained. Due to the degeneracy of the code, considerable changes can occur in nucleotide sequences encoding the same amino acid sequence. Another embodiment is a sequence capable of hybridizing to clone 75 under mildly stringent conditions (50 ° C., 2 × SSC) and hybridizing with a biologically active type II IL-1R polypeptide. Contains soybean or degenerate sequences.

Recombinant type II IL-1R DNA is
Stable integration (by transformation or transduction) of the recombinant transcription unit into the chromosomal DNA, or
Having a recombinant transcription unit as one of the constituents of a resident plasmid, such as E. bacteria such as E. coli and S. It is expressed or amplified in a recombinant expression system that comprises a substantially homogeneous single culture of host microorganisms, such as yeast such as C. cerevisiae. In general,
The cells that make up the line are the progeny of a single progenitor transformant. A recombinant expression system as defined herein will express a heterologous protein by the induction of a DNA sequence to be expressed or a regulatory factor linked to a synthetic gene.

The transformed host cell is a recombinant DNA
Cells transformed or transduced with the type IIIL-1R vector constructed using the technology. Transformed host cells usually express type II IL-1R, but host cells transformed to clone or amplify type II IL-1R DNA need not express type II IL-1R . The expressed type II IL-1R is selected type II I
Depending on L-1R DNA, it is located on the cell membrane but is secreted into the culture supernatant. Suitable host cells for expression of mammalian type II IL-1R include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes are, for example, E. Gram-negative or Gram-positive bacteria such as E. coli or Bacillus are included. Higher eukaryotic cells include established cell lines of mammalian origin, as described below. DNA of the present invention
It is also possible to use the RNA obtained from the construct to produce mammalian type II IL-1R in a cell-free system. Bacterial, fungal, yeast and mammalian host cells have been described by Pouwels et al. (CloningVectors: A Laborat).
ory Manual, Elsevier, New Y
Ork, 1985), the relevant disclosures of which are incorporated herein by reference.

Prokaryotic expression host cells are used for the expression of type II IL-1R, which does not require extensive proteolytic and disulfide reactions. Prokaryotic expression vectors generally include selectable markers with one or more phenotypes, such as genes encoding proteins that confer antibiotic resistance and genes that supplement auxotrophy, and It contains a host-recognized origin of replication that ensures amplification. Prokaryotic host organisms suitable for transformation are E. coli, Bacillus
subtilis, Salmonella typh
imurium and Pseudomonas, Str
eptomyces and Staphylococcu
Various species within the s genus are included, but others are used in the matter of choice.

A useful expression vector for bacteria is the well known cloning vector pBR322 (ATCC37
017), which may include a selectable marker and a bacterial transcription start point derived from a commercially available plasmid containing the genetic element.
Such commercially available vectors are eg pKK2
23-3 (Pharmacia Fine Chemi
cals, Uppsala, Sweden) and pGE
M1 (PromegaBiotec, Madison,
WI, USA). These pBR322 "spine" species combine an appropriate promoter with the structural gene to be expressed. E. coli
Is an E. Escherichia coli is typically transformed with a derivative of pBR322, a plasmid obtained from E. coli (Bolivar et al., Gene 2:95,
1977). pBR322 contains genes for ampicillin and tetracycline resistance, facilitating identification of transformed cells.

Promoters commonly used in recombinant microbial expression vectors include β-lactamase (penicillinase) and lactose promoter systems (Chan et al.
Hang), Nature 275: 615, 1978 and Goeddel et al., Nature 28.
1: 544, 1979) Tryptophan (trp) promoter system (Maniatis, Mo.
regular Cloning: A Laborat
ory Manual, Cold Spring Har
bor Laboratory, p. 412, 198
2) is included. A particularly convenient bacterial expression system utilizes the lambda phage P _L promoter and the cI857 _ts temperature dependent repressor. Vectors available from the American Type Culture Collection that contain derivatives of the λP _L promoter are available from E. coliJMB9
(ATCC37092) strain, pHUB2 plasmid, and E. coli RR1 strain (ATCC5308
It contains the pPLc28 plasmid resident in 2).

Recombinant type II IL-1R protein is expressed in yeast host cells, especially in S. cerevisi
It is also expressed in Saccharomyces species, such as ae. Pichia or Kluyvero
Yeasts of other genera, such as myces, are also utilized.
Yeast vectors are generally derived from the yeast 2μ plasmid, or
It contains an origin of replication derived from an autonomously transcribed sequence (ASR), a promoter, DNA encoding type II IL-1R, a sequence for polyadenylation and transcription termination, and a selection gene. Suitably, the yeast vector contains an origin of replication, and yeast also contains E. E. coli, such as E. E. coli ampicillin resistance gene and S. cerevisiae tryptophan, which is a selectable marker for a mutant strain of yeast that cannot grow in T
It is desirable to include a promoter derived from a highly expressed yeast gene for inducing transcription of the RP1 or URA3 gene and a downstream structural gene. Therefore,
The presence of TRP1 or URA3 lesions in the yeast host cell genome will provide an effective environment for detecting transformation by growth in the absence of tryptophan or uracil.

Suitable promoter sequences in yeast vectors include the promoters of metallothionein, 3-phosphoglycerate kinase (Hitzeman).
n) et al. J. Biol. Chem. 255: 207
3, 1980) or other enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trio. Promoters of glycolytic enzymes such as sephosphate isomerase, phosphoglucose isomerase and glucokinase (Hess et al., J. Ad.
v. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 1
7: 4900, 1978). For suitable vectors and promoters for use in yeast expression systems, see R. et al. R. Hitzeman et al. , EPA7
3, 657.

A preferred yeast vector is E. coli. coli
It can be assembled from the yeast DNA containing the pUC18-derived DNA sequence for selection and replication (Amp ^γ gene and origin of replication), the glucose-repressible ADH2 promoter, and the α-factor secretory leader sequence. Regarding the ADH2 promoter, Russell (Rus
sell) et al., (J. Biol. Chem. 258: 2.
674, 1982) and Beier et al.,
(Nature 300: 724, 1982). The yeast α-factor leader sequence, which promotes the secretion of the heterologous protein, can be inserted between the promoter and the structural gene to be expressed. For example, Kurjan et al., Cell 30: 933, 198.
2; and Bitter et al. , Proc. N
atl. Acad. Sci. USA 81: 5330, 1
See 984. The leader sequence may be modified at its 3'end with one or more restriction enzyme sites facilitating fusion of the leader sequence with the foreign gene. Suitable yeast transformation methods are known to those of skill in the art; typical techniques are Heinnen.
Et al., Proc. Natl. Acad. Sci. USA7
5: 1929, 1978, for selection of Trp ⁺ transformants, 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose, 10μ.
Selection medium containing g / ml adenine and 20 μg / ml uracil; or 0.67% for URA ⁺ transformants
YNB and Sherman et al., Labo
rattle Course Manual for
Methods in Yeast Genetics
s, Cold Spring Harbor Labo
rally, Cold Spring Harbo
r, New York, 1986 using a medium supplemented with amino acids and bases.

The host strain transformed with the vector consisting of the ADH2 promoter is used for expression in an amount of 80 μm.
1% yeast extract, 2% peptone and 1% supplemented with g / ml adenine and 80 μg / ml uracil
Or it could be grown in a rich medium consisting of 4% glucose. The crude yeast supernatant is collected by filtration and kept at 4 ° C until further purification.

Various mammalian or insect cell lines in culture are also advantageously used to express the recombinant protein. Expression of recombinant proteins in mammalian cells is
It is particularly preferred because the protein produced is generally correctly folded, properly modified and fully functional. An example of a suitable mammalian cell line was described by Gluzman (Cell2).
3: 175, 1981), CO from monkey kidney cells.
S-7 strain, and for example L cells, C127, 3T3, Chinese hamster Ovalley (CHO), Hel
a and other cell lines capable of expressing the appropriate vector, including the BHK cell line. Mammalian expression vectors include origins of replication, appropriate promoters and enhancers linked to the gene to be expressed, and nontranscribed elements such as nontranscribed sequences 5 ′ or 3 ′ flanked by ribosome binding sites, polyadenylation sites, It may also contain splicing donor and acceptor sites, and 5'or 3'untranslated sequences such as transcription termination sequences. For the Baculovirus system for producing heterologous proteins in insect cells, see Luckow and Summers.
mmers) review, Bio / Tech
6. 6:47 (1988).

Transcriptional and translational control sequences in the expression vector that will be used to transform the cells of the Beast animal may be provided by the source of the virus. For example, the most commonly used promoters and enhancers are from polyoma, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus. DN derived from SV40 virus genome
A sequences such as the SV40 origin, early and late promoters, enhancers, splicing and polyadenylation sites could be used to supply other genetic elements required for expression of heterologous DNA sequences. Both early and late promoters are SV40
It is particularly useful because it can be easily obtained from a virus as a fragment containing a viral replication origin (Fears (F
iers) et al. , Nature 273: 113, 197.
8). Smaller or larger SV can be obtained if it contains the approximately 250 bp nucleotide sequence from the Hind3 site located at the viral origin of replication to the Bgll site.
It seems that 40 fragments may also be used. In addition, the mammalian genomic type II IL-1R promoter, control, and / or signal sequence may be
Such control sequences may be utilized provided they are compatible with the cells of the chosen host. Further details regarding recombinant mammalian type II IL-1R producing mammalian high expression vectors are provided in Example 2 below. Typical vectors are Okayama and Berg (B
erg) (Mol. Cell. Biol. 3: 280, 19).
83).

A system useful for stable high level expression of mammalian receptor cDNA in C127 mouse mammary epithelial cells is essentially Cosman.
Et al. (Mol. Immunol. 23: 935, 198).
6).

In the advantage of the present invention, Type II I
The recombinant expression vector containing L-1R cDNA is stably integrated into the DNA of the host cell. Elevated levels of expression product are achieved by selecting cell lines in which the number of vector DNA has been amplified. Cell lines in which the number of vector DNAs has been amplified are selected, for example, by transforming host cells with a vector containing a DNA sequence encoding an enzyme that is inhibited by known agents. The vector may also include a DNA sequence encoding the protein of interest. In addition, the host cell can be used as a DN encoding the protein of interest.
A second vector containing the A sequence cotransforms (C
Transform). The transformed or cotransformed host cells are then cultured at increasing concentrations of the known drug, thereby selecting drug resistant cells. Such drug resistant cells often survive increased concentrations of toxic drugs due to overproduction of the drug-inhibited enzyme, often as a result of amplification of the gene encoding the enzyme. When drug resistance is caused by an increase in the copy number of the vector DNA encoding the enzyme to be inhibited, the DN of the host cell is
The protein of interest in A (eg, type IIIL-1
There is a concomitant co-amplification of the vector DNA encoding R).

A more preferred system for such co-amplification uses the gene for dihydrofolate reductase (DHFR) which can be inhibited by the drug metrexate (MTX). To achieve co-amplification, a host cell lacking an active gene encoding DHFR was added to DHF.
Transform with a vector containing R and a DNA sequence encoding the protein of interest, or D
A vector containing a DNA sequence encoding HFR and a vector containing a DNA sequence encoding a protein of interest are cotransformed. Transformed or cotransformed host cells are cultured in medium containing increased levels of MTX to select surviving cell lines.

A particularly preferable co-amplification system is a gene for glutamine synthase (GS) which is involved in synthesizing glutamine by hydrolyzing ATP into ADP and phosphoric acid from glutamic acid and ammonia to promote the reaction. I am using. GS is inhibited by various inhibitors such as methionine sulfoximine (MSX). Therefore, type II IL-1R is transformed into a vector comprising GS and the DNA sequence of the target protein, or a vector containing the DNA sequence encoding GS and a vector containing the DNA sequence encoding the target protein. Co-amplified cells can be co-amplified and cultured at high concentrations by culturing host cells in medium containing increased levels of MSX and selecting for surviving cells. The GS co-amplification system, suitable recombinant expression vectors and cell lines are described in the following PCT application: WO87 / 04462, WO89 / 01036, W.
O89 / 10404 and WO86 / 05807.

Recombinant proteins can be expressed in mammalian cell lines such as Chinese Hamster Ovalley (CHO) cells,
Alternatively, SP2 / O-Ag14 is a mouse myeloma cell line such as NSO or YB2 / 3.0-Ag2
0-like rat myeloma cell line (PCT application WO /
89/10404 and WO 86/05807), DHFR is also preferably expressed by co-amplification of GS.

A more preferred eukaryotic vector for expression of type II IL-1R DNA is described in Example 2 below.
Will be disclosed in. This vector, named pDC406, is derived from the mammalian high expression vector pDC201 and contains regulatory sequences from SV40, HIV and EBV.

Purification of recombinant type II IL-1R Purified mammalian type II by culturing a suitable host / vector system expressing the translation product of the DNA of the present invention and purifying from the culture medium or cell extract. IL-1R or an analog thereof is prepared.

For example, recombinant soluble type II IL
The supernatant in the system that secretes the -1R protein into the medium is first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. obtain. Following concentration, the concentrate is applied to a suitable purification carrier. For example, a suitable affinity carrier comprises IL-1 or a lectin or antibody molecule bound to a suitable support. Alternatively, anion exchange resins such as carriers or substrates with, for example, diethylaminoethyl (DEAE) addition can also be used. As the carrier, acrylamide, agarose, dextran, cellulose or other types commonly used for protein purification can be used. Suitable cation exchangers include a variety of insoluble carriers containing sulfopropyl or carboxymethyl groups. A sulfopropyl group is more preferred.

Finally, one or more RP-HPLCs using a hydrophobic reverse phase high performance liquid chromatography (RP-HPLC) support, eg silica gel with the addition of methyl groups or other aliphatic groups. Can be further performed to purify the type II IL-1R construct.
Some or all of the above-mentioned purification operations can also be used for the preparation of homologous recombinant proteins in various combinations.

Recombinant proteins made from bacterial cultures are usually first isolated by extraction from cell pellets and subjected to one or more concentration, salting out, aqueous phase ion exchange or size exclusion chromatography procedures. Be seen. Finally high performance liquid chromatography (HPLC) may be performed as the final purification operation. Recombinant mammal type I
The cells of the microorganism used for the expression of IL-1R were frozen-
It can be disrupted by any convenient method, including melt cycling, sonication, mechanical disruption, or the use of cell lysing reagents.

Fermentation of yeast expressing soluble mammalian type II IL-1R as a secreted protein greatly simplifies purification. The secreted recombinant protein resulting from large-scale fermentation is described by Urdal et al.
l. ) (J. Chromatog. 296: 171,1)
984) and can be purified by a similar procedure. This reference is a preparative HPLC column with two consecutive columns for purification of recombinant human GM-CSF.
A reverse phase HPLC run is described.

Human type I synthesized in recombinant culture
I IL-1R is a human type II IL-from culture.
It is ranked by the amount and nature of non-human cellular components, including proteins, which depends on the purification procedure to recover 1R. These components are usually of yeast, prokaryotic or non-human higher eukaryotic origin, and are preferably present in amounts of less than about 1% by weight and in amounts of contaminants that are harmless to their presence. Furthermore, recombinant cell culture is usually of the type IIIL- type in natural origin, eg intracellularly, in cell exudates or body fluids.
Type II without the protein associated with 1R
Allows the production of IL-1R.

Of recombinant soluble type II IL-1R
Therapeutic Administration The present invention provides a method of using a therapeutic composition comprising an effective amount of soluble type II IL-1R protein and a suitable diluent and carrier, and the administration of an effective amount of soluble type II IL-1R protein. By a method for suppressing human IL-1 dependent immune response.

For therapy, patients, preferably humans, are administered purified solution type II IL-1R protein by appropriate treatment as indicated. Thus, the soluble type IIIL-1R protein component may be administered by pill injection, continuous infusion, sustained release from implants or other suitable technique. Typically soluble type II
The IL-1R therapeutic agent is administered in the form of a component consisting of the purified protein in association with a physiologically acceptable carrier, excipient or diluent. Such carriers are nontoxic to recipients in the amounts and concentrations used. In general, the preparation of such components is based on Type II IL-1.
R is a buffer, an antioxidant such as ascorbic acid,
With a combination of low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, glucose, carbohydrates including sucrose or dextrin, chelating agents such as EDTA, glutathione and other stabilizers and excipients.
Neutral buffered saline or saline mixed with homologous serum albumin is typically a suitable diluent. Preferably, the product is stylized as a lyophilizate using a suitable excipient solution (eg sucrose) as the diluent. The appropriate dose can be determined by trial. Generally, shuIL-
1R dose is about 1 mg / kg / day to about 10 mg / kg
/ Day, preferably about 500 μg / kg / day to about 5 m
It is expected to induce physiological effects at g / kg / day.

IL-1R-1 and Type II IL-1R
Since both proteins bind IL-1R, similar, if not identical, soluble type II IL-1R proteins would be expected to have therapeutic activity. For example, soluble human type II IL-1R can be administered, eg, for the purpose of suppressing an immune response in humans. A wide variety of diseases and conditions result from immune responses to alloantigens, including allograft rejection and graft-versus-subject reactions. In the alloantigen-induced immune response, shuIL-1R suppresses lymphocyte proliferation and inflammation leading to T cell activation. Therefore s
huIL-1R is an immunity induced by alloantigens in clinical treatments such as allografts (such as skin, kidney, heart transplants) as well as graft-subject reactions in patients undergoing bone marrow transplants. It is used to effectively suppress the reaction.

Soluble human type II IL-1R is also used in the clinical treatment of autoimmune dysfunctions such as rheumatoid arthritis, diabetes and multiple sclerosis that depend on T cell activation for antigens that are not recognized as self-specific. Can be used.

The following examples are given by way of illustration and not limitation.

[0069]

Example 1 Direct activation of active protein in CV-1 / EBNA-1 cells
Encodes human type II IL-1R by direct expression
Isolation of cDNA Radiolabeled recombinant human IL-1 of rIL-1β
β is represented by Kronheim et al.
l. , E. coli (E. col.
It was prepared by purifying so that it was expressed in i) and in the same room.
(Bio / Technology 4: 1078, 198
6) IL-1β with di-iodo ( ¹²⁵ I) Bolton-hunter reagent (New
England Nuclear, Glenolde
n, PA). 10 ul of phosphoric acid (0.01
5 mol / L) buffer salt solution (PBS: 0.15 mol / L)
L), 10 micrograms (0.
57 nmol of protein was added to sodium borate (0.
1 mol / L) -buffer salt solution (0.15 mol / L),
It was mixed with pH 8.5, 10 uL and reacted with Bolton-Hunter reagent 1 mCi (0.23 nmol) for 12 hours at 8 ° C. according to the manufacturer's instructions. Next, 2% gelatin 30
uL and 1 mol / L glycine ethyl ester were added and the protein was loaded in a 1 mL volume Biogel ^™ P6 column (Bio-Rad Laboratories, Ri.
chmond, CA) and separated from unreacted Bolton-Hunter reagent. There was always 50% to 60% label binding. Iodine label is 1 × 10 ¹⁵ to 5
× 10 ¹⁵ cpm / mmol-1 (0.4 to 2 atoms I
A specific activity of 17.5 kD was detected by sodium dodecyl sulfate-polyacrylamide gel electromigration (SDS-PAGE), and the molecular weight was IL.
Consistent with the previously reported value of -1. The labeled protein was TCA-precipitable by 98% or more, suggesting that I ¹²⁵ was covalently bound to the protein.

B. Creation of CB23 cDNA library
And screening SV40, human immunodeficiency virus (HIV) and Epstein-Barr (Epstein)
-Barr) monkey kidney cell line CV-1 / EB of cDNA clones collected using a mammalian expression vector (pDC406) containing regulatory sequences of the virus (EBV)
NA-1 (the CV-1 cell line was
The CB23 library was generated and screened by direct expression (from those transformed with the gene encoding -1). The CV-1 / EBNA-1 cell line constitutively expresses EBV nuclear antigen-1 under the control of an enhancer / promoter that operates from the immediate early stage of human cytomegalovirus (CMV), and thus has an EBV origin of replication. Allows autonomous replication of expression vectors such as pDC406. The expression vector used was pDC406, which was derived from HAV-EO and used by Dower et al.
weret al. J. Immunol, 142: 4.
314, 1989), which in turn is p
Derived from DC201, high level expression is possible in the CV-1 / EBNA-1 cell line. pDC406 is HAV-E
It differs from O (Dower et al., Supra) in that there is no intron in the tripartite leader sequence of type 2 adenovirus in HAV-EO.

The CB23 cDNA library is generally described by Ausubel et al., E.
ds. , Current Protocols in
Molecular Biology, Vol. 1,1
987 to isolate poly (A) from total RNA extracted from B-cell lymphothymic CB23 as described in 987.
⁺ MRNA produced by reverse transcription (Benjamin & Dower, Bloo
d75: 2017,1990). CB23 cell line is EB
V infectious cord blood cell (CB) lymphoid cell line, which is described in Benjamin et al. Proc.
Natl. Acad. Sci. USA 81: 3547,
Derived using the method described in 1984. Poly (A) ⁺
The mRNA was separated by oligo dT cellulose chromatography and was roughly analyzed by Gubler and Huffman (Gubler).
and Hoffman), Gene 25: 263,
Double-stranded cDNA was prepared as described in 1983. Briefly, poly (A) ⁺ mRNA was converted to RNA-cDNA hybrids by reverse transcriptase using random 6 nucleotides as primers. Then D
Using RNAase H in collaboration with NA polymerase I,
The RNA-cDNA hybrid was changed to a double-stranded cDNA. The resulting double-stranded cDNA was blunt-ended with T4 DNA polymerase. Reversed double non-phosphorylated oligonucleotide was annealed and the resulting blunt end cDNA
Blunt end ligation with the ends of A and DNA ligase was performed by Haymerle et al. Nucl.
eic Acids Research, 14: 861
5, 1986.

[0072]

In this case, it is expected that only the 24-mer oligo will ligate to the cDNA. Unligated oligos were removed by gel filtration chromatography at 68 ° C, leaving a 24-nucleotide non-self-complementary overhang on the cDNA. Using the same method, the mammalian expression vector pDC406SalI-cleaved 5'end was cDNA
Was changed to a 24 nucleotide overhang complementary to the one added in. The vector with the adapter and the cDNA were ligated at the optimum ratio in the presence of T4 polynucleotide kinase. The ligation mixture is dialyzed into E. coli strain D
It was electroporated into H5α. About 3.9 x 10
^Six clones were generated and plated out in approximately 3000 populations. Samples of each population were used for frozen glycerol storage and were also used to obtain populations of plasmid DNA.
The collected DNA was then transfected into monkey CV1 / EBNA-1 cell pseudo-confluent cell layers using DEAE-dextran and described by Ruthman et al.
an et al. , Nucl. Acids Res.
11: 1295 (1983) and McCutchan et al., J. Natl.
Chloroquine (Chlo, similar to that described in Cancer Inst, 41: 351 (1986).
roquine) treatment was performed. CV-1 / EBNA-
One cell was derived as follows. CV-1 / EB
The NA-1 cell line structurally expresses EBV nuclear antigen-1 which is regulated by the immediate early enhancer / promoter of CHV. African Green Monkey Kidney Cell Line CV-1 (ATCC CCL70) contains 5 μg of pSV
gpt [Mulligan & B (Mulligan & B
erg), Proc. Natl. Acad. Sci. U
SA78: 2072,1981] and 25 μg of pDC3
03 / EBNA-1 with calcium phosphate coprecipitation technique (Aus-Bell et al. Eds., Current Protocol)
ols in Molecular Biology,
Wiley, New York, 1987) was co-transfected. pDC303 / EBNA-1 is p
DC302 [Mosley et a
l. ), Cell 59: 335, 1989]. First, the introns present in the Atez virus 3rd part leader sequence were ligated with the following pair of synthetic oligonucleotides from PvuII to S that span the introns.
The caI fragment was removed by exchanging it to create plasmid pDC303.

[0074]

Next, the Epstein-Barr virus nuclear antigen I (EBNA-1) was coded and the EBV map 10
HindII which is originally from 7,932 to 109,894
I-AhaII restriction fragment (Baer et al.
al. ), Nature 310: 207, 1984).
Was then inserted into the multiple cloning site of pDC303 to create plasmid pDC303 / EBNA-1. Transfected cells were grown in the presence of hypoxanthine, aminopterin, thymidine, xanthine and mycophenolate according to standard methods (Ausberg et al., Supra; Mulligan and Burke supra) and transfected plasmids Stably contaminated cells were selected. The resulting drug resistant colonies were isolated and expanded into each cell line for analysis. EBNA-1 functioning cell line
Were screened by the expression of One cell line clone 68 was found to express EBNA-1 using this assay and was designated CV-1 / EBNA-1.

To transfect CV-1 / EBNA-1 cells with the cDNA library, the cells were cultured in complete medium (Dulbecoo's Modified Eagle (E).
agle) medium (DMEM) 10% (v / v) placental bovine serum (FCS), 50 U / ml penicillin, 50 U / m
1 streptomycin, containing 2 mM L-glutamine)
, And plated in 6-well dishes (Falcon) or 1-well chambered slides (Lab-Tek) at a density of 2 × 10 ⁵ cells / well. 1 ml human fibronectin (10 μg / ml P
After pretreatment for 30 minutes in PBS, the cells were washed once with PBS. The medium was removed from the adherent cell layer and replaced with 1.5 ml complete medium containing 66.6 μM chloroquine sulfate. 0.2 ml D
NA solution (2 μg DNA in complete medium containing chloroquine,
0.5 mg / ml DEAE-dextran) was then added to the cells and incubated for 5 hours. After the incubation, the medium was removed and the cells were shocked by the addition of complete medium containing 10% DMSO for 2.5 to 20 minutes, then the solution was replaced with fresh complete medium. The cells were allowed to grow in culture so that the inserted sequences could be expressed. CV-1 / EBNA- that survives under these conditions
A transfection efficiency of 80% of 1 cell was obtained.

After 48 to 72 hours, the transfected CV-1 / EBNA cell monolayers were examined for binding of radioiodinated IL-1β prepared as described above by slide autoradiography. Binding protein expression was assayed. Transfected CV-1 / EBNA-1 cells with binding medium (RP
MI medium 1640, 25 mg / ml bovine serum albumin, 2 mg / ml sodium azide, 20 mM HEPE
Wash once with S, pH 7.2 and 50 mg / ml non-fat dry milk (NFDM)), 3 × 10 ^-9 M ¹²⁵ I
-Incubated with 1 ml binding medium containing IL-1β ⁺ NFDM for 2 hours at 4 ° C. After incubation, the cells in the chamber slide are combined with binding buffer + NFDM
Rinse 3 times with PBS, then 2 times with PBS, pH 7.3, unbound
¹²⁵ I-IL-1β was removed. Cells are PBS, pH 7.
3. Incubate in 10% glutaraldehyde for 30 minutes at room temperature to fix, wash twice with PBS and air dry. Slides were dipped in Kodak GTNB-2 photographic emulsion (6x diluted with water) and exposed in the dark for 48 hours to 7 days in the dark. Slide the slides to Kodak D19 Developer (40
Develop with g / 500ml water for about 5 minutes, rinse with water,
It was fixed with a faG433C fixer. Each slide was examined under a microscope at 25-40 × magnification and tested for type II IL-1.
Positive cells expressing R were fixed by the presence of autoradiographic silver particles in a bright background.

Cells in 6-well dishes were washed once in binding buffer + NFDM and then 3 times with PBS pH 7.3 to remove unbound ¹²⁵ I-IL-1β. Bound cells were then trypsinized and removed from the plate and bound ¹²⁵
I-IL-1β was counted by a beta counter.

Using the slide autoradiography method, the population of about 3000 cDNA was 250,0
Only after screening the 00 cDNA, the assay of transfectant populations clearly showed many cells positive for IL-1β binding. This population was then divided into 500 populations and screened again by slide autoradiography to identify one positive population. The population was further divided into 75 groups, plated in 6-well plates and screened by a plate binding assay which was analyzed by quantification of bound ¹²⁵ I-IL-1β. Cells were scraped from the plates and counted to determine which 75 populations were positive. Individual colonies of this 75 population were screened and one clone (clone 75) was identified as carrying the synthesis of a protein detectable in the IL-1β binding assay. This clone was isolated and the insert was sequenced to sequence human type II IL-1R cDNA clone 75. Human type II IL-1R cDNA clone 7
PDC406 cloning vector with 5 has pHuI
Named L-1R-II 75 American Type Culture Collection (ATCC) (Rockv
ille MD, USA) on June 5, 1990 under deposit number CRL10478. Nucleic acid (SEQ ID NO: 1) and predicted amino acid sequence of clone 75 (SEQ ID NO: 1 and SEQ ID NO: 2)
And the sequence listing which published the relevant information is given later in this specification.

[0080]

Example 2 cDNA encoding human soluble type II IL-1R
And full-length type IL-1RcDN in expression vector pDC406
CDNA encoding soluble human type II IR-1R by polymerase chain reaction (PCR) amplification using A clone 75 (SEQ ID NO: 1) as a template.
(With a sequence of amino acids 13-333 of SEQ ID NO: 1). The following 5'oligonucleotide primers (SEQ ID NO: 7) and 3'oligonucleotide primers (SEQ ID NO: 8) were first made.

[0081]

The 5'primer is human type II IL-
This nucleotide sequence corresponds to human clone 75 nucleotides 31-51, since it corresponds to the 31-51 nucleotides of the untranslated region of 1R clone 75 (SEQ ID NO: 1) with a SalI restriction enzyme site at the 5'end. It can anneal to the complementary (-) strand. The 3'primer was labeled with nucleotides 1191 to 1172 (human type II IL-1R clone 75 (SEQ ID N
O: 1) consisting of an anti-sense nucleotide encoding 3 amino acids) and a NotI restriction enzyme site added at 5'and carrying a stop codon.

The following PCR reagents were added to a 1.5 ml Eppendorf microcentrifuge tube: 10 × PCR buffer (500 mM KCl, 100 mM Tris-HCl, p.
H8.3, 25 mM 15 mM MgCl ₂ and 1 mg /
ml gelatin) (Perkin-Elmer Cetus; Per
kin-Elmer Cetus, Norwalk, C
N) 10 μl, 2 mM solution containing each dNTP (2 mM
dATP, 2 mM dcTP, 2 mM dGTP and 2 mM
dTTP) 10 μl, Taq DNA polymerase 2.
5 units (500 units 1 ml solution 0.5 μl)
(Perkin-Elmer Cetus), template DNA 50 ng
And to each 5 μl of the above oligonucleotide primer 20 μM solution was added 74.5 μl of water to make a final volume of 100 μl and the final mixture was topped with 100 μl of paraffin oil. First, denature the template at 94 ° C for 90 seconds, and
Reannealed at 5 ° for 75 seconds and cDN at 72 ° C for 150 seconds
A is extended to a DNA temperature circulator (Erycomp (Er
PCR was performed using icomp) San Diego CA). Step program (94 °, 25 seconds denaturation, 5
Amplification using 5 °, 45 sec annealing, 72 ° 150 sec extension) followed by 20 additional cycles of PCR followed by 7
It was extended at 2 ° for 5 minutes.

The sample was deparaffinized by phenol-chloroform extraction and span column chromatography on G-50 (Boehringer Mannheim) to remove D.
NA was extracted. Extracted DNA 10 μl fraction with 1% Sea
Kem ^™ Agarose (FMC BioProduct
s, Rockland, ME)
Staining with ethidium bromide confirmed that the DNA fragment size was identical to the expected product.

Then, 20 μl of PCR-amplified cDNA
The product was digested with SalI and NotI restriction enzymes using standard methods. The SalI / NotI restriction enzyme fragment was then lysed with 1.2% Seaplaque ^™ low temperature (LGT).
After separation on agarose, the band representing the fragment was isolated. The fragment was ligated into pDC406 by standard in-gel ligation method and the vector was CV1.
-Transfected into EBNA cells and expressed as described above in Example 1.

[0086]

Example 3 of cDNA encoding mouse type II IL-1R
The isolated mouse type II IL-1R cDNAs were cloned from a cDNA library prepared from mouse precursor B cell line 7OZ / 3 (ATCCTIB158) into human type II
It was isolated by interspecific hybridization using the IL-1R probe. A cDNA library was constructed in a λ phage vector by in vitro packaging (Gigapack, Stratagene, San Diego) using the λgt10 arm according to the manufacturer's instructions. Two
The full-chain human type II IL-1R probe is approximately 1.35 kb Sa of human type II IL-1R clone 75.
The lI restriction enzyme fragment was cut out and the cDNA was prepared using random primers (Boehringer Mannheim).
Was labeled with ³² P. The mouse cDNA library was amplified once and a total of 5 × 10 ⁵ plaques were screened with human probe in 35% formamide (5 × SSC, 42 ° C.). Some mouse type II
An IL-1R cDNA clone (including clone λ2) was isolated, but none of the clones appeared to be full length. Using the cleotide sequence information obtained from the partial clones, full-length mouse type II IL was obtained as follows.
The -1R cDNA was cloned.

A full-length cDNA clone encoding mouse type II IL-1R was cloned from Flowman et al.
an et al. ) Proc. Natl. Acad.
Sci. USA 85: 9898
It was isolated using the mouse precursor B cell line 70Z / 3 by the A-terminal rapid amplification (RACE) method. Simply put, RAC
Method E uses PCR to amplify a copy of the cDNA in the region between the known end of the cDNA transcript (determined from the nucleic acid sequence obtained above) and the three ends. An adapter primer having a sequence containing a 17 dT base pair and an adapter sequence containing three endonuclease recognition sites (convenient restriction enzyme sites were added to the three ends of cDNA) was used as mRN.
Used for reverse transcription of the A population and generation of (-) strand cDNA. Mouse type II IL-1R clone 2c described above
A primer complementary to a known stretch of the sequence of the 5'untranslated region of DNA and oriented in the 3'direction is the (-) strand cDNA.
Anneal with NA and extend to generate complementary (+) strand cDNA. The resulting double-stranded cDNA was converted to the original 5 '
Amplify by PCR using primers that anneal to the ends and the synthetic 3'end poly (A) tail. The details of the RACE method are as follows.

The following PCR oligonucleotide primers (respectively d (T) ₁₇ adapter primer, 5 '
The amplification primer and the 3'amplification primer) were first made.

[0089]

Briefly, the d (T) ₁₇ adapter primer (SEQ ID NO: 9) contains a nucleic acid sequence that anneals to the poly (A) ⁺ region of the mRNA transcripts of the population, and the Used to generate reverse transcripts. DNA amplified by PCR
It contains endonuclease restriction enzyme sites for PstI, NatI and BamHI introduced in 5'amplification primer (SEQ ID NO: 10) is mouse type II
It corresponds to a 5'-untranslated region of IL-1R clone λ2 with a SalI restriction enzyme site added 5'to nucleotides 15-34, and this nucleotide sequence was generated by reverse transcription with a d (T) ₁₇ adapter primer (- ) Annealed to the strand cDNA and extended to produce a (+) strand cDNA.
The 3'primer (SEQ ID NO: 11) was annealed with the (+) strand DNA having the above endonuclease restriction site and extended to produce a double-stranded full-length cDNA encoding mouse type II IL-1R, which was Therefore, it can be amplified by a normal PCR reaction. Details of the PCR method are as follows.

Maniatis et al.
al. ) Molecular Cloning: A
Laboratory Manual (Cold Sp
ring Harbor 1ab. , NY, 1982).
Poly (A) ⁺ mRNA was isolated by oligo dT cellulose chromatography from total RNA extracted from 70Z / 3 cells using the standard method described in (3) and reverse transcribed as follows. About 1 μg of poly (A) ⁺ mRNA in 16.5 μl water was heated at 68 ° C. for 3 minutes and then cooled on ice, and 2 μl of 10 × RTC buffer (500 m
M Tris-HCl at 22 ° C., pH 8.7 60 mM
MgCl ₂ 400 mM KCl 10 mM DTT,
10 units of RNasin for each dNTP)
(Promega Biotech; Promega Biote
ch) 0.5 μg of d (T) ₁₇ -adapter primer and 10 units of AMV reverse transcriptase (Life Science; Life Sciences) in a total amount of 20 μl
In addition, mRNA was reverse transcribed by incubation at 42 ° C. for 2 hours to synthesize a cDNA population. The reaction mixture is TE
Buffer (10 mM Tris-HCl, pH 7.
(5, 1 mM EDTA) and left overnight at 4 ° C.

About 1 or 5 μl of the cDNA population was added to 5 μl of a 20 μM solution of 5 ′ amplification primer containing a sequence corresponding to the nucleotide 15-34 sequence of mouse type II IL-1R clone λ2, 5 μl of a 3 μm amplification primer 20 μM solution; 10 μl of 10 × PCR buffer (50
0 mM KCl, 100 mM Tris-HCl (p
H8 ・ 4,20 ℃), 14mM MgCl ₂ and 1mg /
ml gelatin), 4 μl of 5 mM each dNTP (5 mMd
ATP, 5mMdCTP, 5mMdGTP, 5mMdT
TP), 2.5 units (standard 5000 units / ml solution 0.5 μl), Taq DNA polymerase (Perkin-Elmer Cetus Instrument (I
nstrument) and diluted to a volume of 100 μl and mixed. The final mixture was overlaid with 100 μl paraffin oil. First, the template was denatured at 94 ° C. for 90 seconds, reannealed at 64 ° and 75 seconds, the cDNA was extended at 72 ° and 150 seconds, and PCR was performed using a DNA temperature circulator (Perkin-Elmer / Cetus). . PCR was performed for 25 additional cycles of amplification using a step program (94 °, 25 sec denaturation, 55 °, 45 sec annealing, 72 °, 150 sec extension), then 7 ° at 72 °.
Minute final extension was performed.

Extract the sample with phenol-chloroform and G
DNA was removed by span column chromatography at -50 (Boehringer Mannheim) to remove paraffin oil.
Was extracted. Extracted DNA 10 μl fraction with 1% SeaKe
Separation by electrophoresis on m ^™ agarose (FMC Bioproduct Rockland, ME) and staining with ethidium bromide confirmed that the DNA fragment size was identical to the expected product. The gel was then blotted and the mouse type II IL-1R clone λ2 561 described above was used.
Using the 0 bp EcoRI fragment as a probe, it was confirmed that the band contained DNA encoding mouse type II IL-1R.

The PCR-amplified cDNA product was then concentrated by centrifugation in an Eppendorf microcentrifuge at maximum speed for 20 minutes, followed by ethanol precipitation in 1/10 volume sodium acetate (3M) and 2.5 volume ethanol. 30 μl of the concentrate was digested with SalI and NotI restriction enzymes using standard methods. SalI / NotI restriction enzyme fragment
The band corresponding to the fragment was isolated by separation on a 1.2% LGT agarose gel. The restriction enzyme fragment was then transferred from agarose to Gene Clean ^™ (Bio-101, La.
Purified from Jolla, CA). The resulting purified restriction enzyme fragment was ligated into the pDC406 vector, which was transfected into CV-1EBNA cells and expressed as described above in Example 1. Spread (S
EQ IDNO: 12) and predicted amino acid sequence (SEQ
The sequence listing, which published ID NO: 12 and SEQ ID NO: 13) and related information, is given later in this specification.

[0095]

Example 4 cDN Encoding Mouse Soluble Type II IL-1R
Preparation and expression of A Soluble mouse type II IL- was prepared by PCR amplification using 70Z / 3 poly (A) ⁺ mRNA as a template and the following method described for the full-length clone encoding mouse type II IL-1R. CDNA encoding 1R
(Having the sequence of amino acids 13-345 of SEQ ID NO.12) was created. The following PCR oligonucleotide primers (each d (T) ₁₇ adapter primer,
5'amplification primer and 3'amplification primer) were prepared.

[0096]

The d (T) ₁₇ adapter primer and the 5'amplification primer are described in Example 5 and SEQ ID
It is the same as D NO: 9 and SEQ ID NO; 10. The 3'end of SEQ ID NO: 12 is SEQ ID
It is complementary to nucleotides 1145-1166 of NO: 12 and has a 5'addition to the NotI restriction enzyme site and a stop codon.

Using the d (T) ₁₇ adapter primer as described in Example 3, poly (A) ⁺ mRNA to c
A DNA population was synthesized. In a 1.5 ml Eppendorf microcentrifuge tube, about 1 μl of the cDNA population, 5 μl of 5 μl amplification primer 20 μM solution, 5 μl of 3 ′ amplification primer 20 μM solution, 10 μl of 10 × PCR buffer (500 mM kCl, 100 mM Tris-HC
l (pH 8.4 at 20 ° C.), 14 mM MgCl ₂ and 1 mg / ml gelatin) 10 μl 5 mM each dNTP
4 μl 2.5 units (including 5 mM dATP, 5 mM dCTP, 5 mM dGTP and 5 mM dTTP) 2.5 units (standard 500 units / ml solution 0.5 μl) TaqD
NA polymerase (Perkin-Elmer Cetus Instrument) was mixed and diluted with 75.4 μl of water to a volume of 100 μl. The final mixture was overlaid with 100 μl paraffin oil. First, the template was denatured at 94 ° C. for 90 seconds, reannealed at 55 ° and 75 seconds, the cDNA was extended at 72 ° and 150 seconds, and PCR was performed using a DNA temperature circulator (Elycop). Step program (9
4 °, 25 seconds denaturation, 55 °, 45 seconds annealing, 72
PCR was performed for an additional 25 cycles, followed by a final extension at 72 ° for 7 minutes.

The sample was subjected to phenol-chloroform extraction and span column chromatography on G-50 (Boehringer Mannheim) to remove paraffin oil and DN.
A was extracted. Extracted DNA 10 μl fraction with 1% Sea
Separation by electrophoresis on Kem ^™ agarose (FMC Bioproduct Rockland, ME) and staining with ethidium bromide confirmed that the DNA fragment size was identical to the expected product.

The PCR amplified cDNA product was then concentrated by centrifugation in an Eppendorf microcentrifuge at maximum speed for 20 minutes,
Ethanol precipitation was then carried out in 1/10 volume sodium acetate (3M) and 2.5 volume ethanol. 50 μl of the concentrate was digested with SalI and NotI restriction enzymes using standard methods. SalI / NotI restriction enzyme fragment
The band corresponding to the fragment was isolated by separation on a 1.2% LGT agarose gel. The restriction enzyme fragment was then purified from the isolated band using the freeze-thaw method described below. The band from the gel was split into two 175 μl fragments and placed in a 1.5 ml Eppendorf microcentrifuge tube. 500 μl of separation band (0.15 M NaCl 10 mM Tri
s, pH 8.0 1 mM EDTA) was added to each tube and the tubes were heated at 68 ° C. until the gel melted. Then freeze the gel on dry ice for 10 minutes, thaw at room temperature,
It was centrifuged at 0 ° C. for 30 minutes. The supernatant is then removed, transferred to a new tube, suspended in 2 mL ethanol, and further suspended at 30 ° C at 4 ° C.
Centrifugation was performed to cause DNA precipitation. DNA precipitation 70
% Ethanol was removed from the centrifuge tube for 5 minutes, and resuspended in 20 μl TE buffer.

The resulting purified restriction enzyme fragment was then pD
It was ligated to the C406 vector. Ligation samples were transformed into DH5α and colonies were analyzed to check for the correct plasmid. The vector was transfected into COS-7 cells and expressed as described above in Example 1.

[0102]

Example 5 Type II IL-1R Binding Studies The binding inhibition constants of recombinant human type II IL-1R expressed and purified as described in Example 1 above were determined by varying concentrations of competitor (IR). -1β or IL-1α) was incubated with a fixed amount of radiolabeled IL-1β or IL-1α and cells expressing type II IL-1R to determine the inhibitory binding assay. The competitor binds to the receptor and blocks the radiolabeled ligand from binding to the receptor. Binding assays are generally performed by Doher et al., J. Am. Immuno
l. 132: 751, 1984 and Park et al. (Park
et al. ) J. Biol. Chem. 261: 41
77, 1986 by the phthalic acid oil separation method. Briefly, CV1 / EBNA cells were placed in 6-well dishes (Costar Cambridge, MA) for 2 hours at 4 ° C with ¹²⁵ I-IL-1β and 1 m.
l-Binding medium (Rose Well Park Memorial Institute (RPMI) 1640 medium with 2% BS
A, 20 mM Hepes buffer containing 0.2% sodium azide pH 7.2).
Sodium azide is included at 37 ° C. to prevent ¹²⁵ I-IL-1 from being taken up and degraded by cells. The plates were incubated for 1 hour at 37 ° C on a rotary shaker. Then, a plurality of fractions of the incubated mixture were transferred to a polyethylene centrifuge tube containing a phthalic acid oily mixture having a ratio of 1.5 dibutylphthalic acid and 1 bis (S-ethylhexal) phthalic acid. 100
A control tube containing x molar excess of unlabeled IL-1β was also included to determine non-specific binding. Union
¹²⁵ I-IL-1 cells containing ¹²⁵ I-IL-1 were subjected to eppendorf microcentrifugation at 15,000 × g for 5 minutes to unbound ¹²⁵ I-
Separated from IL-1. Radioactivity bound to cells was determined with a gamma counter. This assay (type II IL
Inhibiting binding of ¹²⁵ I-IL-1β to −1R using unlabeled human IL-1β as a competitor), full-length human type II IL-1R has a K _I1 of about 19 ± 8 ×
10 ⁹ and K _I2 are about 0.2 ± 0.002 × 10 ⁹ 2
It was suggested to show a phasic binding to IL-1β. ¹²⁵
Using unlabeled human IL-1β to inhibit the binding of I-IL-1α to type II IL-1R, full-length human type II IL-1R has a K _I1 of about 2.
0 ± 1 × 10 ⁹ and K ₁₂ are about 0.013 ± 0.003
It showed a biphasic binding to IL-1β of × 10 ⁹ .

The binding inhibition constants of soluble human type II IL-1R expressed and purified as described in Example 2 above, show that various concentrations of IL-1β competitor were radiolabeled with a fixed amount of I-. It was determined by an inhibitory binding assay that was incubated with CB23 cells (Epstein-Barr virus transformed code blood cell B lymphocyte line) expressing IL-1β and type II IL-1R. The binding assay is also done by Dower et al. J. I
mmunol 132: 751, 1984 and Park et al. Biol. Chem. 261: 4177 by the phthalic acid oily separation method. Briefly, COS-7 cells were transformed into soluble human type I as described above.
PDC40 containing cDNA encoding IL-1R
6 expression vectors were transfected. Supernatants from COS cells were cultured for 3 days after transfection, followed by binding medium (2% BSA in Rosewell Park Memorial Institute (RPMI) 1640 medium,
50 mM in 20 mM Hepes buffer and 0.2% sodium azide pH 7.2 in a 6-well dish.
Diluted to a volume of μl / well. 50 of supernatant
μl of 9 × 10 ^-10 M ¹²⁵ I-IL-1β and 2.5 × 10
⁶ Incubated with CB23 cells at 8 ° C. for 2 hours with shaking. Two 60 μl fractions of the incubation mixture were transferred to a polyethylene centrifuge tube containing a phthalic acid oily mixture consisting of dibutylphthalic acid 1.5, bis (S-ethylhexal) phthalic acid 1 ratio. A negative control tube containing 3 × 10 ⁻⁶ M unlabeled IL-1β was also included to determine non-specific binding (100% inhibition) and a positive control tube containing 50 ml binding medium with only radiolabeled IL-1β was used. Included to determine maximum binding. Combined ¹²⁵ I-
Eppendorf microcentrifuge cells with IL-1 15,
¹²⁵ I-IL-unbound after centrifugation at 000 × g for 5 minutes
Separated from 1. The supernatant containing unbound ¹²⁵ I-IL-1β was discarded and the cells were carefully rinsed with ice-cold binding medium. Cells are then trypsin-EDTA 1 ml
The cells were cultured by incubating at 37 ° C for 15 minutes.
Radioactivity of cells was determined by gamma counter. This assay ( ¹²⁵ I-IL-1β binding to soluble human type II
Inhibiting with IL-1R) showed that soluble human type II IL-1R has a K _I of about 3.5 × 10 ⁹ M ⁻¹ . Soluble human type I using a similar method
Inhibition of IL-1α binding by I IL-1R
Soluble human type IIIL-1R is 1.4 × 10 ⁸ M ⁻¹
It was shown to have a K _I of.

Mouse type II IL-1R is K
2 less than _I1 0.8 × 10 ⁹ and K _I2 0.01 × 10 ⁹
It showed binding to phase IL-1β.

[0105]

Example 6 Type II IL-1R Affinity Crosslin
King Research Affinity Cross Linking Research, Park et al., P
roc. Natl. Acad. Sai. USA 84:
It was performed as described in 1669 1987. The recombinant human IL-1α and IL-1β used in this assay were expressed and purified and labeled as described previously. (Dower et al., J. Exp. Med. 1
62: 501, 1985; Dower et al., Nature.
324: 266, 1986), a recombinant human IL-1 receptor antagonist (IL-1ra) was synthesized by Eisenberg et al. Nature.
343: 341, 1990)
Cloned with the sequence and transiently transfected and expressed in COS cells, Dower et al.
Immunol. 143: 4314,1989, soluble human type bound to affinity gel.
Purified by affinity chromatography on a column of IL-1R and eluted at low pH.

Briefly, recombinant type II IL-
CV1 / EBNA cells expressing 1R (4 × 10 ⁷
/ Ml) to ¹²⁵ I-IL-1α or ¹²⁵ I-IL-1β
(1 nM) and 4 ° C, 1 μM as specificity control
Were incubated for 2 hours in the presence and absence of excess unlabeled IL-1. Then wash the cells,
Bis (sulfosuccinimidyl) suberate was added to a final concentration of 0.1 mg / ml. After placing at 25 ° C for 30 minutes, the cells were washed, 2 mM leupeptin, 2 mM0
-Phenanthroline, and 100 μl of phosphate buffered saline solution containing 2 mM EGTA to prevent proteolysis (P
Resuspended in BS) / 1% Triton. Equal amount (CP
The extracted supernatant fraction containing the specificity control equivalent to ¹²⁵ I-IL-1 of M) was ^subjected to S on a 10% gel using standard techniques.
It was analyzed by DS / PAGE.

FIG. 4 shows native and recombinant mouse and human type IIL-1 versus natural controls performed as described above with radiolabeled IL-1α and IL-1β. Figure 2 shows the results of an affinity cross-linking study for the size comparison of the receptor and recombinant mouse and human type II IL-1 receptor proteins. Usually the recombinant protein is probably CV1
/ As a result of the difference in sugar chain type when overexpressed in EBNA cells, the size of the recombinant receptor that transiently migrates as a slightly faster band and slightly wider band is similar to that of the natural receptor. The results also showed that the type II IL-1 receptor was smaller than the type I IL-1 receptor. Certain combinations (IL-1β and native human type I receptor) did not give rise to specific cross-linking products. Since approximately equal amounts of label were loaded on each experimental lane, this combination was strongly expected to be relatively weakly cross-linked, as indicated by the intensity of the unbound ligand band at the bottom of the gel. It ¹²⁵ I-IL-1
Natural human type II IL-1 cross-linked with α
Lanes showing receptor-expressing cells show the size range of the complex between native and recombinant type I receptor (Mr = 10).
The compositional component appears in 50,000). Such a complex is not detected in lanes containing recombinant type II IL-1 receptor, but probably because CB23 cells contain traces of type I IL-1 receptor mRNA.
This may be the result of low level expression of the type I IL-1 receptor in CB23 cells.

[0108]

Example 7 Preparation of Type II IL-1R Monoclonal Antibody Purified recombinant type II IL-1R, eg human type II IL-1R or type II IL-1.
Preparation of transfected COS cells expressing high levels of R was performed and performed using conventional techniques, eg US Pat.
Monoclonal antibodies against type II IL-1R are made using techniques such as those disclosed in US Pat. No. 1,993. This antibody shows that IL-1 is type II
IL in that it interferes with binding to L-1R, for example, in improving toxicity and other undesirable effects of IL-1
-1 or soluble type II IL-1R is expected to be useful as a component of diagnostic and research assays.

To immunize mice, type II IL
-1R Immunogen Complete Freund (Fre, und's)
Suspended in adjuvant, 10-1
Balb / c mice were injected subcutaneously intraperitoneally in an amount of 00 μg width. After 10-12 days, the immunized animals were boosted with a booster in suspension in Incomplete Freund's Adjuvant and then periodically on a 1 to 2 week immunization schedule. Serum samples are taken periodically by posterior orbital bleeding or caudal incision and examined by dot blot assay (antibody sandwich) or ELISA (enzyme linked immunosorbent assay) or receptor binding inhibition. Other assay methods are applicable. Following detection of the appropriate antibody titer, positive animals are injected intravenously with the antigen in saline. After 3-4 days, the animals were sacrificed, spleen cells were cultured and the mouse myeloma cell line NS1 or Ag8.65 was used.
Fuse with 3. The hybridoma cell line obtained by this method is plated on multiple microtiter plates in HAT selection medium (hypoxanthine, aminopterin, thymidine) to prevent the growth of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma clones thus prepared were tested for reactivity with type II IL-1R, for example, Engval et al. Immun.
ochem. 8: 871 (1971) and U.S. Pat.
It is possible to screen by ELISA by applying the technique disclosed in the specification of US Pat. No. 703,004. High concentrations (> 1 mg / ml) of anti-type II IL were injected by intraperitoneal injection of positive clones and into syngeneic Balb / c mice.
Ascites fluid containing the -1R monoclonal antibody is prepared or grown in a flask or a rotating bottle. The resulting monoclonal antibody was subjected to ammonium sulfate precipitation followed by gel filtration chromatography,
And / or Staphylococcus aureus (Stap
hylococuus aureus) protein A
Alternatively, it can be purified by affinity chromatography based on the binding of an antibody to Protein G of Streptococcus.

[0111]

[Simple description of sequence listing] SEQ ID NO: 1 and SE
Q ID NO: 2 shows the nucleotide sequence and predicted amino acid sequence of human type II IL-1R. The mature peptide encoded by this sequence was defined as amino acids 1-385. The predicted signal peptide is amino acid-
It is defined as 13 to -1. The predicted transmembrane region is designated as amino acids 331-335.

SEQ ID NO: 3-SEQ ID
NO: 6 is the various oligonucleotides used to clone the full length human type II IL-1R.

SEQ ID NO: 7 to SEQ ID
NO: 8 is an oligonucleotide primer used to make soluble human type IIIL-1R by polymerase chain reaction (PCR).

SEQ ID NO: 9-SEQ ID
NO: 11 is full-length and soluble mouse type II IL
Oligonucleotide primers used to clone -1Rs.

SEQ ID NO: 12 and SEQ I
D NO: 13 is full length mouse type II IL-1R
The nucleotide sequence and predicted amino acid sequence of are shown. The mature peptide encoded by this sequence has amino acids 1-3
It was set at 97. The predicted signal peptide was defined as amino acids -13 to -1. The predicted transmembrane region is designated as amino acids 343-368.

SEQ ID NO: 14 is an oligonucleotide primer used to make soluble mouse type II IL-1R.

[0117]

[Sequence list]

 (1) Information of SEQ ID NO: 1 (i) Properties of sequence (A) Length: 1357 base pairs (B) Type: Nucleic acid (C) Strand: 2 (D) Form: Linear (ii) Molecular type : CDNA for mRNA (iii) Hypothesis: N (iv) Antisense: N (vi) Source of origin (A) Organism: Homo sapiens (G) Cell type: Human B-cell lymphocytic line (H) Cell line: CB23 (vii ) Direct origin (A) Library: CB23 cDNA (B) Clone: pHuIL-1RII75 (ix) Features (A) Name / Key: CDS (B) Position: 154. ． 1350 (D) Other information: (ix) Features: (A) Name / key: mat peptide (B) Position: 193. ． 1347 (D) Other information: (ix) Features: (A) Name / key: sig peptide (B) Position: 154. ． 192 (D) Other information: (xi) Sequence description: SEQ ID NO: 1: Sequence

[0118]

[0119]

(1) Information of SEQ ID NO: 2 (i) Nature of sequence: (A) Length: 398 amino acids (B) Type: amino acid (D) Form: linear (ii) Molecular type: protein (xi) ) Sequence description: SEQ ID NO: 2: Sequence

[0121]

[0122]

(1) Information of SEQ ID NO: 3 (i) Sequence properties: (A) Length: 24 base pairs (B) Type: Nucleic acid (C) Strand: 1 (D) Form: Linear ( ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Sequence description: SEQ ID NO: 3: Sequence TCGACTGGAA CGAGACGACC TGCT 24 (1) SEQ ID NO: 4 Information (i) Sequence properties: (A) Length: 20 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear (ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: Y (xi) Sequence description: SEQ ID NO: 4: Sequence GACCTTGCTC TGCTGGACGA 20 (1) SEQ ID NO: 5 information (i) Sequence properties: ( ) Length: 46 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear (ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Sequence description: SEQ ID NO: 5: Sequence CTGTTGGGCT CGCGGTTGAG GACAAACTCT TCGCGGTCTT TCCAGT 46 (1) SEQ ID NO: 6 information (i) Sequence nature: (A) Length: 46 base pairs (B) Type: Nucleic acid (C) Chain: One (D) Form: Linear (ii) Molecular type: DNA (Genome) (iii) Hypothetical: N (iv) Antisense: Y (xi) Sequence description: SEQ ID NO: 6: Sequence GACAACCCGA GCGCCAACTC CTGTTTGAGA AGCGCCAGAA AGGTCA 46 (1) SEQ ID Information of NO: 7 (i) Sequence properties: (A) Length: 29 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear (ii) Molecular type: DNA (genome ) (Iii) Assumption: N (iv) Antisense: N (xi) Description of sequence: SEQ ID NO: 7: Sequence GCGTCGACCT AGTGGACGCTC ATACAAATC 29 (1) Information of SEQ ID NO: 8 (i) Nature of sequence: (A) ) Length: 33 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear (ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Description of sequence: SEQ ID NO: 8: Sequence GCGCGGCCCGC TCAGGAGGAG GCTTCCCTTGA CTG 33 (1) Information of SEQ ID NO: 9 (i) Nature of sequence: (A) Length: 37 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear (ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N ( xi) Sequence description: SEQ ID NO: 9: Sequence CTGCAGGCGGG CCGCGGATCC TTTTTTTTTTTTTTTTTT 37 (1) SEQ ID NO: 10 information (i) Sequence properties: (A) Length: 28 base pairs (B) Type: nucleic acid (C) Chain: 1 strand (D) Form: Linear (ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Sequence description: SEQ ID NO: 10 : Sequence GCGTCGACGG CAAGAAGCAG CAAGGTAC 28 (1) Information of SEQ ID NO: 11 (i) Properties of sequence: (A) Length: 20 base pairs (B) Type: Nucleic acid (C) Chain: One (D) Form: Linear (ii) Molecular type: DNA (Genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Sequence description : SEQ ID NO: 11: Sequence CTGCAGGGCGG CCGCGGATCC 20 (1) Information of SEQ ID NO: 12 (i) Sequence properties: (A) Length: 1366 base pairs (B) Type: Nucleic acid (C) Strand: 2 (D) Morphology: Linear (ii) Molecular type: cDNA for mRNA (iii) Hypothetical: N (iv) Antisense: N (vi) Root origin (A) Organism: Mouse (H) Cell line: 70Z / 3 (vii) direct origin (A) library: 70Z / 3 (B) clone: 12 (ix) features: (A) name / key: CDS (B) position: 85. ． 1317 (D) Other information: (ix) Features: (A) Name / key: mat peptide (B) Position: 124. ． 1314 (D) Other information: (ix) Features: (A) Name / key: sig peptide (B) Position: 85. ． 123 (D) Other information: (xi) Sequence description: SEQ ID NO: 12: Sequence

[0124]

[0125]

(1) Information of SEQ ID NO: 13 (i) Properties of sequence: (A) Length: 410 amino acids (B) Type: amino acid (D) Form: linear (ii) Molecular type: protein (xi ) Sequence description: SEQ ID NO: 13: Sequence

[0127]

[0128]

(1) Information of SEQ ID NO: 14 (i) Nature of sequence: (A) Length: 39 base pairs (B) Type: nucleic acid (C) Strand: 1 (D) Form: linear ( ii) Molecular type: DNA (genome) (iii) Hypothetical: N (iv) Antisense: N (xi) Sequence description: SEQ ID NO: 14: Sequence GCGCGGCCGC CTAGGAAGAG ACTTTTTGA CTGTGG 36

[Brief description of drawings]

FIG. 1 is a schematic diagram of the expression plasmid pDC406.
The cDNA molecule inserted at the Sal site is transcribed and translated using the regulatory factors obtained from HIV and adenovirus. pDC406 is SV40, Epstein-Barr virus and pBR3
Contains the origin of replication from 22.

FIG. 2 is a schematic representation of the human and mouse type II IL-1 receptor and various human and mouse clones used for sequencing. Thin lines indicate untranslated regions and coding regions are drawn as squares. The portion encoding the signal peptide is filled in. The transmembrane portion is shaded by crossed diagonal lines. And intracellular proteins are stippled. The potential N-glycosylation site is indicated by an inverted triangle. Immunoglobulin-like disulfide bonds are shown by the dotted lines connecting the two sulfur molecules (S ... S).

FIG. 3 Amino acid sequences of human and mouse type II IL-1 receptors (obtained from cDNA clones)
For human and mouse type I IL-1 receptor amino acid sequences (Sims et al., Proc. Natl.
Acad. Sci. USA 86: 8946,1989;
Sims et al., Science 241: 585, 198.
8) and the amino acid sequence of the ST2 cell gene (Tominaga, FEBS Lett. 258; 301, 1989).
And B15R open reading frame of Vassinia virus (Smith and C
han), J. Gen. Virology 72:51
1, 1991). Numbering starts with the starting methionine. The predicted position of signal peptide cleavage in each sequence is von Heijne,
Nucl. Acids. Res. 14: 4683, 19
86) and determined by the method described in (86), and is indicated by a space between a portion expected to be a signal peptide and the body of the protein. The predicted transmembrane and intracellular regions of the type II IL-1 receptor are shown at the bottom, separated by a gap. Residues conserved in all four IL-1 receptor sequences are shown in black on a black background. Residues conserved in each other's type II receptors are shaded and type IIIL-1 of each other is shaded.
Residues that are conserved in the receptor are boxed. Cysteine residues forming disulfide bonds characteristic of immunoglobulin structure are marked with a black circle and
The set of two extra cysteines at the IIL-1 receptor, as well as some cysteines in other sequences, are marked with an asterisk. Appropriate boundaries for domains 1, 2 and 3 are shown above each row. The predicted signal peptide cleavage at the type II IL-1 receptor is after Ala13,
It produces an unusual short signal peptide, which results in an N-terminus that is 12 (human) or 23 (mouse) amino acids longer than the corresponding position of the N-terminus of the human or mouse type I IL-1 receptor after cleavage. Mouse type II IL
Another less preferred but possible cleavage site for the -1 receptor is after Thr15 or Pro17. This arrangement is done by hand and does not show the objective optimal arrangement of arrangements. Human and mouse type II
The full length and soluble nucleotide and amino acid sequences of the IL-1 receptor cDNA are also given in the sequence listing therein.

FIG. 4: SDS / P of cross-linked IL-1 receptor
It is an autoradiograph of AGE gel. Cells expressing the IL-1 receptor are cognate unlabeled I
¹²⁵ I-IL-in the absence or presence of L-1 competitor
Cross-linked with 1, extracted, electrophoresed, Example 6
The autoradiograph was done as described in. Recombinant receptor was transiently expressed in CV1 / EBNA cells.
The cell line used for cross-linking with the natural receptor is KB (ATCCCCL1717) (human type I
IL-1R), CB23 (human type II IL-1)
R), EL4 (ATCC TIB39) (for murine type IIL-1R) and 70Z / 3 (ATCC TIB)
158) (for murine type II IL-1R).

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12N 5/10 C12P 21/02 C 9282-4B G01N 33/53 P // C12Q 1/68 A 9453-4B ( C12P 21/02 C12R 1:91) 8412-4B C12N 5/00 B (72) Inventor David John Cosman, Washington 9898 United States, 98110, Northeast Ewing Street, Northeast Ewing Street 10129 (72) Inventor Steven Dee Rupton Washington State 98115 USA, Theatre, Sandpoint Way North East 7323, number 211 (72) Inventor Bruce Mosley United States Washington 98125, Theater, Thirty Sixth Avenue North East 11311 (72) Inventor Steven Kay Dewar Washington, USA 98052 , Redmont, East Lake Summit Parkway Wow North East 2620

Claims (15)

[Claims] 1. A biologically active type II IL
An isolated DNA sequence encoding the -1 receptor (type II IL-1R) protein. 2. A cDNA clone having a nucleotide sequence derived from a region encoding a native type II IL-1R gene; (b) a mild stringent condition (50 ° C., 2 × SSC) in the clone. And a biologically active type I
A DNA sequence encoding the I IL-1R protein; and (C) a type II IL-which is degenerate and biologically active as a result of the genetic code to the DNA sequence defined in (a), (b). DNA encoding 1R protein
The isolated DNA sequence of claim 1, selected from the group consisting of: a sequence; 3. The isolated DNA sequence of claim 1, which encodes a soluble type II IL-1R protein. 4. The soluble type II IL-1R protein is selected from the group consisting of a sequence of amino acid residues 1-133 of SEQ ID NO: 1 and a sequence of amino acid residues 1-345 of SEQ ID NO: 12. An isolated DNA sequence according to claim 3. 5. D according to any one of claims 1-4
A recombinant expression vector containing an NA sequence. 6. A method for preparing a biologically active mammalian type II IL-1 receptor, which comprises culturing a suitable host cell containing the vector of claim 5 under conditions that promote expression. 7. A type II with homogeneous biological activity.
IL-1 receptor (type II IL-1R) protein. 8. A soluble human type II IL-1R protein with homogeneous biological activity. 9. A sequence of amino acid residues 1-333 of SEQ ID NO: 1 and SEQ ID NO: 1 of 1
A homogeneous biologically active type II IL-1R protein according to claim 7 selected from the group consisting of a sequence of -345 amino acid residues. 10. An effective amount of type II according to claim 7.
A pharmaceutical composition suitable for parenteral administration to a human patient for controlling an immune response, comprising an IL-1R protein and a suitable diluent or carrier. 11. To control an immune response in a mammal,
Use of type II IL-1R protein in the preparation of a medicament. 12. The use of claim 11, wherein the type II IL-1R protein is human type II IL-1R and the mammal treated is human. 13. Type I in the preparation of a medical composition suitable for parenteral administration to human patients for the control of immune responses.
Use of I IL-1R protein. 14. (a) Mammalian type I to IL-1 molecule
Binds an IL-1 receptor protein; and (b)
Type II IL-comprising the step of detecting or measuring the amount of unbound IL-1 or type II IL-1 receptor
A method for detecting 1 or type II IL-1R molecules or their interactions. 15. An antibody that immunoreacts with and has specificity for a mammalian type II IL-1 receptor.